The goal of this research project, which is highly responsive to NIH/NIDA RFA-DA-11-007, is to develop a high throughput screening (HTS) assay for screening and identifying selective ligands for neuronal nicotinic acetylcholine receptors (nAChRs) containing a3, a5, a6 and/or b4 subunits. nAChRs are ligand-gated ion channels that are implicated in a wide range of physiological functions, pathological processes and pharmacological effects. They have been shown to mediate the addictive effects of nicotine, which are the main reason why more than a billion people worldwide smoke cigarettes or use other tobacco products. Neuronal nAChRs are composed of a and b subunits that form pentameric cation channels. Nine a subunits (a2 - a10) and three b subunits (b2 - b4) have been identified in vertebrate neuronal tissues. Although the a7 subunit forms functional homomeric receptors, most nAChRs in mammalian brain are heteromeric receptors that contain two or more subunits. For many years, a4b2 nAChR subtypes (composed of a4, b2 and perhaps other subunits) have been the primary focus for studying nicotine addiction and developing smoking cessation therapeutics, and much evidence supports their involvement;but since 2007 genome-wide association studies have revealed significant associations of markers in the human a5-a3-b4 gene cluster on chromosome 15q24-25.1 with smoking and smoking related diseases. Among these markers, the most compelling variant is rs16969968, which changes an amino acid from aspartate to asparagine at position 398 (D398N) in the a5 subunit. This and more recent discoveries suggest that in addition to a4 and b2 subunits, a3, a5, a6 and b4 subunits play important roles in smoking and smoking related diseases. In light of this new information, it is considered important to develop new ligands selective for receptors containing a3, a5, a6 and/or b4 subunits. Hence, the crucial need for a HTS assay that can reliably identify selective ligands in existing or new libraries of compounds. We propose two Specific Aims to accomplish this goal: Aim 1: To establish highly functional stably transfected cell lines that express human nAChRs containing a3, a5, a6 and/or b4 subunits. Aim 2: To develop a functional HTS assay using IonFlux automated, high throughput patch clamp system. PUBLIC HEALTH RELEVANCE: Cigarette smoking is the leading cause of preventable disease and premature death in the United States. Selective ligands of nicotinic receptors can be used in studying nicotine addiction and for developing new smoking cessation therapeutics. The goal of this project is to develop high throughput screening assay that will accelerate the development of selective ligands.